In Article <Pine.3.87.9408291250.D1876-0100000 at csuvax1>,
obrien at CSUVAX1.MURDOCH.EDU.AU (Philip OBrien) wrote:
>Dear Netters
>We have been trying unsuccessfully to do northern blots of fungal RNA.
>The RNA when electrophoresed on formaldehyde gels shows intense bands on
>rRNA and they appear to be intact. The RNA is not degraded. We have
>both vacuum blotted and capillary blotted using 10XSSC as the transfer
>buffer. We have also dot blotted using 10XSSC and denaturing the RNA in
>formaldehyde in the presence of formamide. The membranes we have used
>were Hybond N, and N+. We usually load about 2-10ug RNA. The dot blots
>also contained DNA controls. The only hybridization we have seen is to
>the DNA controls so obviously our probe is labelled, and we should see
>something when we use amplified rDNA probes since a large proportion of
>the RNA should be rRNA. However we see nothing. I would like to ask
>people who have experience of northerns, what is the most appropiate
>transfer buffer, and membrane to use. Grateful for any suggestions.
>>Philip O'Brien
>BES, Murdoch University
>Western Australia
>obrien at csuvax1.murdoch.edu.au>
Phillip,
I am guessing you are not getting efficient transfer. How do you monitor
this? For Northerns, I always use downward transfer with 50mM NaOH, and get
good transfer within two hours. I normally load more than 10ug of RNA also,
up to 35ug in some cases. I have been using Bio-Rad Zeta-probe GT membranes
for about a year, and find them to be good membranes, with a very high
binding capacity, although the background is high on some lots. I also stain
my gels prior to transfer by including EtBr in the gel-loading buffer. This
has not presented any problems for me, and it allows me to monitor the
efficiency of the transfer of the rRNA bands. Good luck.
Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu